Outlook for Cellulase Improvemente (Zhang Y.-h. P. Et Al., 2006)

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Research review paper

Outlook for cellulase improvement: Screening and selection strategies

Y.-H. Percival Zhang a,, Michael E. Himmel b, Jonathan R. Mielenz c

a Biological Systems Engineering Department, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061, USAb National Bioenergy Center, National Renewable Energy Laboratory, Golden, CO 80401, USA

cLife Science Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831, USA

Received 31 January 2006; received in revised form 6 March 2006; accepted 11 March 2006

Available online 27 March 2006

Abstract

Cellulose is the most abundant renewable natural biological resource, and the production of biobased products and bioenergy

from less costly renewable lignocellulosic materials is important for the sustainable development of human beings. A reduction in

cellulase production cost, an improvement in cellulase performance, and an increase in sugar yields are all vital to reduce the

processing costs of biorefineries. Improvements in specific cellulase activities for non-complexed cellulase mixtures can be

implemented through cellulase engineering based on rational design or directed evolution for each cellulase component enzyme,

as well as on the reconstitution of cellulase components. Here, we review quantitative cellulase activity assays using soluble and

insoluble substrates, and focus on their advantages and limitations. Because there are no clear relationships between cellulase

activities on soluble substrates and those on insoluble substrates, soluble substrates should not be used to screen or select

improved cellulases for processing relevant solid substrates, such as plant cell walls. Cellulase improvement strategies based on

directed evolution using screening on soluble substrates have been only moderately successful, and have primarily targeted

improvement in thermal tolerance. Heterogeneity of insoluble cellulose, unclear dynamic interactions between insoluble substrate

and cellulase components, and the complex competitive and/or synergic relationship among cellulase components limit rational

design and/or strategies, depending on activity screening approaches. Herein, we hypothesize that continuous culture using

insoluble cellulosic substrates could be a powerful selection tool for enriching beneficial cellulase mutants from the large library

displayed on the cell surface.

2006 Elsevier Inc. All rights reserved.

Keywords: Cellulase activity assay; Cellulose; Cellulosome; Continuous culture; Enzymatic cellulose hydrolysis; High throughput screening;

Selection; Sugar assay

Biotechnology Advances 24 (2006) 452481

www.elsevier.com/locate/biotechadv

Abbreviations:AFEX, ammonia fiber explosion; BC, bacterial cellulose; BCA, 2,2-bicinchroninate; BMCC, bacterial microcrystalline cellulose;

CMC, carboxymethyl cellulose; CBM, cellulose-binding module; CBP, consolidated bioprocessing; CrI, crystallinity index; DMAc, N,N-

dimethylacetamide; DNS, dinitrosalicyclic acid; DP, degree of polymerization of cellulose; DS, degree of substitution; DTT, dithiothreitol; Fa,

fraction of-glucosidic bond accessible to cellulase; FPA, filter paper activity; FRE, fraction of the reducing end to all anhydroglucose units of

cellulose, 1/DP; HEC, hydroxyethyl cellulose; PASC, phosphoric acid swollen cellulose; RAC, regenerated amorphous cellulose; PAHBAH, 4-

hydroxybenzoylhydrazine;RS, selection ratio; TNP-CMC, trinitrophenyl-carboxymethyl cellulose. Corresponding author. Tel.: +1 540 231 7414; fax: +1 540 231 3199.

E-mail address:[email protected](Y.-H. Percival Zhang).

0734-9750/$ - see front matter 2006 Elsevier Inc. All rights reserved.doi:10.1016/j.biotechadv.2006.03.003
mailto:[email protected]://dx.doi.org/10.1016/j.biotechadv.2006.03.003http://dx.doi.org/10.1016/j.biotechadv.2006.03.003mailto:[email protected]


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Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 453

2. Cellulose hydrolysis mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 455

3. Substrates for cellulase activity assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 457

3.1. Soluble substrates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 458

3.2. Insoluble substrates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 458

4. Quantitative assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 460

4.1. Hydrolysis products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 461

4.2. Cellulase activity assays. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 462

4.2.1. Endoglucanases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 462

4.2.2. Exoglucanases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 463

4.2.3. -D-glucosidases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 464

4.2.4. Total cellulase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 464

5. Cellulase improvement and screening/selection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 465

5.1. Rational design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 465

5.2. Directed evolution. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 467

5.3. Screening . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 469

5.4. Selection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4706. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 472

Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 472

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 472

1. Introduction

Cellulose is the primary product of photosynthesis in

terrestrial environments, and the most abundant renew-

able bioresource produced in the biosphere (100

billion dry tons/year) (Holtzapple, 1993; Jarvis, 2003;Zhang and Lynd, 2004b). Cellulose biodegradation by

cellulases and cellulosomes, produced by numerous

microorganisms, represents a major carbon flow from

fixed carbon sinks to atmospheric CO2 (Berner, 2003;

Falkowski et al., 2000; Melillo et al., 2002), is very

important in several agricultural and waste treatment

processes (Angenent et al., 2004; Das and Singh, 2004;

Haight, 2005; Hamer, 2003; Humphrey et al., 1977;

Russell and Rychlik, 2001; Schloss et al., 2005; van

Wyk, 2001), and could be widely used to produce

sustainable biobased products and bioenergy to replacedepleting fossil fuels (Angenent et al., 2004; Demain et

al., 2005; Galbe and Zacchi, 2002; Hall et al., 1993;

Hoffert et al., 2002; Kamm and Kamm, 2004; Lynd,

1996; Lynd et al., 1991, 2002, 1999; Mielenz, 2001;

Mohanty et al., 2000; Moreira, 2005; Reddy and Yang,

2005; Wyman, 1994, 1999, 2003). Additionally, studies

have shown that the use of biobased products and

bioenergy can achieve zero net carbon dioxide emission

(Demain, 2004; Demain et al., 2005; Hoffert et al.,

2002; Lynd et al., 1991, 1999). Development of

technologies for effectively converting less costly

agricultural and forestry residues to fermentable sugars

offers outstanding potential to benefit the national

interest through: (1) improved strategic security, (2)

decreased trade deficits, (3) healthier rural economies,

(4) improved environmental quality, (5) technology

exports, and (6) a sustainable energy resource supply

(Angenent et al., 2004; Caldeira et al., 2003; Demain etal., 2005; Hoffert et al., 1998, 2002; Kamm and Kamm,

2004; Lynd, 1996; Lynd et al., 1991, 1999, 2002;

Moreira, 2005; Wirth et al., 2003; Wyman, 1999).

Effective conversion of recalcitrant lignocellulose

to fermentable sugars requires three sequential steps:

(1) size reduction, (2) pretreatment/fractionation, and

(3) enzymatic hydrolysis (Wyman, 1999; Zhang and

Lynd, 2004b). One of the most important and

difficult technological challenges is to overcome the

recalcitrance of natural lignocellulosic materials,

which must be enzymatically hydrolyzed to producefermentable sugars (Chang et al., 1981; Demain et

al., 2005; Fan et al., 1982; Grethlein, 1984; Hsu,

1996; Lin et al., 1981; McMillian, 1994; Millett et

al., 1976; Moreira, 2005; Mosier et al., 2005; Saddler

et al., 1993; Weil et al., 1994; Wyman, 1999; Wyman

et al., 2005a).

Cellulases are relatively costly enzymes, and a

significant reduction in cost will be important for their

commercial use in biorefineries. Cellulase-based strat-

egies that will make the biorefinery processing more

economical include: increasing commercial enzyme

volumetric productivity, producing enzymes using

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cheaper substrates, producing enzyme preparations with

greater stability for specific processes, and producing

cellulases with higher specific activity on solid

substrates. Recently, the biotechnology companies

Genencor International and Novozymes Biotech have

reported the development of technology that hasreduced the cellulase cost for the cellulose-to-ethanol

process from US$5.40 per gallon of ethanol to

approximately 20 cents per gallon of ethanol (Moreira,

2005), in which the two main strategies were (1) an

economical improvement in production of cellulase to

reduce US$ per gram of enzyme by process and strain

enhancement, e.g., cheaper medium from lactose to

glucose and alternative inducer system and (2) an

improvement in the cellulase enzyme performance to

reduce grams of enzyme for achieving equivalent

hydrolysis by cocktails and component improvement(Knauf and Moniruzzaman, 2004). But this claim has

not yet been widely accepted because the cellulase

mixture was tested only for the specific pretreated

lignocellulosic substrate and cannot be applied to other

pretreated lignocelluloses.

Currently, most commercial cellulases (including -

glucosidase) are produced by Trichoderma species and

Aspergil lus species (Cherry and Fidantsef, 2003;

Esterbauer et al., 1991; Kirk et al., 2002). Cellulases

are used in the textile industry for cotton softening and

denim finishing; in the detergent market for color care,

cleaning, and anti-deposition; in the food industry formashing; and in the pulp and paper industries for de-

inking, drainage improvement, and fiber modification

(Cherry and Fidantsef, 2003; Kirk et al., 2002). The

cellulase market is expected to expand dramatically

when cellulases are used to hydrolyze pretreated

cellulosic materials to sugars, which can be fermented

to commodities such as bioethanol and biobasedproducts on a large scale (Cherry and Fidantsef, 2003;

Himmel et al., 1999; van Beilen and Li, 2002). For

example, the potential cellulase market has been

estimated to be as high as US$400 million per year if

cellulases are used for hydrolyzing the available corn

stover in the midwestern United States (van Beilen and

Li, 2002). This market scenario represents an increase of

33% in the total US industrial enzyme market

(Wolfson, 2005). The large market potential and the

important role that cellulases play in the emerging

bioenergy and bio-based products industries provide agreat motivation to develop better cellulase preparations

for plant cell wall cellulose hydrolysis. These improved

cellulases must also have characteristics necessary for

biorefineries, such as higher catalytic efficiency on

insoluble cellulosic substrates, increased stability at

elevated temperature and at a certain pH, and higher

tolerance to end-product inhibition.

Fig. 1 shows that cellulase engineering for non-

complexed cellulase systems contains three major

research directions: (1) rational design for each

cellulase, based on knowledge of the cellulase structure

and the catalytic mechanism (Schulein, 2000; Wilson,2004; Wither, 2001); (2) directed evolution for each

Rational Design Directed Evolution

endos

exosR

expsNR

-Gase

Screen or select on

solid substrate

Improved cellulase

components

Reconstitute

cellulase

cocktail

Wild type

Cellulase Components

Fig. 1. Scheme of cellulase engineering for non-complexed cellulases. Endos, endoglucanases; exosR, exoglucanases acting on reducing ends;exosNR, exoglucanases acting on non-reducing ends; -Gase,-glucosidase.

454 Y.-H. Percival Zhang et al. / Biotechnology Advances 24 (2006) 452481


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cellulase, in which the improved enzymes or ones with

new properties were selected or screened after random

mutagenesis and/or molecular recombination (Arnold,

2001; Cherry and Fidantsef, 2003; Hibbert et al., 2005;

Schmidt-Dannert and Arnold, 1999; Shoemaker et al.,

2003; Tao and Cornish, 2002); and (3) the reconstitutionof cellulase mixtures (cocktails) active on insoluble

cellulosic substrates, yielding an improved hydrolysis

rate or higher cellulose digestibility (Baker et al., 1998;

Boisset et al., 2001; Himmel et al., 1999; Irwin et al.,

1993; Kim et al., 1998; Sheehan and Himmel, 1999;

Walker et al., 1993; Wilson and Walker, 1991; Zhang

and Lynd, 2004b). With respect to engineering com-

plexed cellulase systems (cellulosomes), the idea of

chimeric constructs of cellulosomal domains/compo-

nents was proposed by Bayer et al. (1994), and the

reconstruction of cellulosome components is becominganother hot research area (Fierobe et al., 2001, 2002,

2005; Mingardon et al., 2005; Sabathe and Soucaille,

2003), which we do not review here.

The cornerstone of enzyme engineering is to achieve

a direct correlation between the enzyme assays or

screening approaches and the changes in enzyme

functions in the desired application. Development of a

useful, predictive cellulase assay or screening is

particularly difficult because of the nature of solid

heterogeneous substrates, such as plant cell walls.

Available quantitative cellulase assays and screenings

have been analyzed and compared herein, includingtheir advantages and limitations. Also, successful

cellulase examples using directed evolution are exam-

ined, and a possible strategy of combinatorial molecular

breeding and continuous culture with solid cellulosic

materials to select a cellulase with higher activity is

discussed.

2. Cellulose hydrolysis mechanisms

Cellulose is a linear condensation polymer consisting

of D-anhydroglucopyranose joined together by -1,4-glycosidic bonds with a degree of polymerization (DP)

from 100 to 20,000 (Krassig, 1993; O'Sullivan, 1997;

Tomme et al., 1995; Zhang and Lynd, 2004b).

Anhydrocellobiose is the repeating unit of cellulose.

Coupling of adjacent cellulose chains and sheets of

cellulose by hydrogen bonds and van der Waal's forces

results in a parallel alignment and a crystalline structure

with straight, stable supra-molecular fibers of great

tensile strength and low accessibility (Demain et al.,

2005; Krassig, 1993; Nishiyama et al., 2003; Notley et

al., 2004; Zhang and Lynd, 2004b; Zhbankov, 1992).

The cellulose molecule is very stable, with a half life of

58 million years for-glucosidic bond cleavage at 25

C (Wolfenden and Snider, 2001), while the much faster

enzyme-driven cellulose biodegradation process is vital

to return the carbon in sediments to the atmosphere

(Berner, 2003; Cox et al., 2000; Falkowski et al., 2000;

Schlamadinger and Marland, 1996).The widely accepted mechanism for enzymatic

cellulose hydrolysis involves synergistic actions by

endoglucanase (EC 3.2.1.4), exoglucanase or cellobio-

hydrolase (EC 3.2.1.91), and -glucosidase (EC

3.2.1.21) (Henrissat, 1994; Knowles et al., 1987;

Lynd et al., 2002; Teeri, 1997; Wood and Garica-

Campayo, 1990; Zhang and Lynd, 2004b). Endoglu-

canases hydrolyze accessible intramolecular -1,4-

glucosidic bonds of cellulose chains randomly to

produce new chain ends; exoglucanases processively

cleave cellulose chains at the ends to release solublecellobiose or glucose; and -glucosidases hydrolyze

cellobiose to glucose in order to eliminate cellobiose

inhibition. These three hydrolysis processes occur

simultaneously as shown in Fig. 2. Primary hydrolysis

that occurs on the surface of solid substrates releases

soluble sugars with a degree of polymerization (DP) up

to 6 into the liquid phase upon hydrolysis by

endoglucanases and exoglucanases. The enzymatic

depolymerization step performed by endoglucanases

and exoglucanases is the rate-limiting step for the

whole cellulose hydrolysis process. Secondary hydro-

lysis that occurs in the liquid phase involves primarilythe hydrolysis of cellobiose to glucose by -glucosi-

dases, although some -glucosidases also hydrolyze

longer cellodextrins (Zhang and Lynd, 2004b). During

cellulose hydrolysis, the solid substrate characteristics

vary, including (1) changes in the cellulose chain end

number resulting from generation by endoglucanases

and consumption by exoglucanases (Kleman-Leyer et

al., 1992, 1994, 1996; Kongruang et al., 2004;

Srisodsuk et al., 1998; Zhang and Lynd, 2005b) and

(2) changes in cellulose accessibility resulting from

substrate consumption and cellulose fragmentation(Banka et al., 1998; Boisset et al., 2000; Chanzy et

al., 1983; Din et al., 1991, 1994; Halliwell and Riaz,

1970; Lee et al., 1996, 2000; Saloheimo et al., 2002;

Walker et al., 1990, 1992; Wang et al., 2003;

Woodward et al., 1992). The combined actions of

endoglucanases and exoglucanases modify the cellu-

lose surface characteristics (topography) over time,

resulting in rapid changes in hydrolysis rates.

The complicated interactions among endogluca-

nases, exoglucanases, and the changing substrate

characteristics during hydrolysis have been simulated

by a new functionally based mathematical model



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(Zhang and Lynd, in press), applying a set of enzymatic

parameters for endoglucanase I, cellobiohydrolases I

and II to a variety of substrates with two important

substrate properties: the fraction of-glucosidic bondaccessible to cellulase (Fa) (Zhang and Lynd, 2004b)

and the degree of polymerization (DP) (Okazaki and

Moo-Young, 1978; Zhang and Lynd, 2004b) (see

Table 1). In this way, disparate information from the

literature was framed in a coherent way to facilitate an

understanding of enzymatic cellulose hydrolysis. For

example, the reaction rates simulated by the model

were consistent with a substantial number of observa-tions reported in the literature, including the effects of

substrate characteristics on exoglucanase and endo-

glucanase activities; the effects of substrate character-

istics and experimental conditions on the degree of

Table 1

Summary of typical values of model celluloses for crystallinity index (CrI), the fraction of-glucosidic bond accessible to cellulase (Fa), which is

estimated by maximum cellulase adsorption capacity (Zhang and Lynd, 2004b), the number average of degree of polymerization (DPN), the fraction

of reducing ends (FRE), and relative ratio of FRE/Fa

Substrates CrI Fa(%)

DPN FRE (%) FRE/Fa

Low High Low High

Soluble

Cellodextrins and their derivatives N.A. 100 26 16.67 50 0.167 0.5

CMC N.A. 100 1002000 0.05 1 0.0005 0.01

Insoluble

Cotton 0.80.95 0.2 10003000 0.033 0.1 0.167 0.5

Whatman No. 1 filter paper 0.45 1.8 7502800 0.036 0.133 0.0198 0.0741

Bacterial cellulose 0.80.95 6 6002000 0.05 0.167 0.00833 0.0278

Microcrytalline cellulose (Avicel) 0.50.6 0.6 150500 0.2 0.667 0.333 1.11

PASC 0 12 1001000 0.1 1 0.00833 0.0833

Pulp (Solka Floc) 0.40.7 1.8 7501500 0.067 0.133 0.0370 0.0741

Pretreated cellulosic substrates 0.40.7 0.6 4001000 0.1 0.25 0.167 0.417

Liquid

Phase

(primary

hydrolysis)

(secondary

hydrolysis)

-Gase

Solid

Phase

endos

exosR

exosNR

Fig. 2. Mechanistic scheme of enzymatic cellulose hydrolysis by Trichodermanon-complexed cellulase system.

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endo-exo synergy; the effects of endoglucanase

partition coefficient on the hydrolysis rates; and the

effects of enzyme loading on relative reaction rates for

different substrates. The model also suggests that it is

nearly impossible to predict hydrolysis performance of

cellulase mixtures from one solid substrate to anothersolid substrate, because of large variations in total

cellulase concentration, ratio of endo/exocellulases,

reaction time, and substrate characteristics. Therefore,

enzyme reconstitution may have be conducted so as to

achieve better performance for a specific substrate

(Knauf and Moniruzzaman, 2004).

Unlike non-complexed fungal cellulase, anaerobic

microorganisms possess complexed cellulase systems,

called cellulosomes (Bayer et al., 1994, 1998, 2004;

Beguin and Alzari, 1998; Demain et al., 2005; Doi

and Kosugi, 2004; Doi et al., 1998; Doi and Tamaru,2001; Leschine, 1995; Schwarz, 2001). Leschine

(1995) estimated that anaerobic cellulose degradation

could account for only 510% of total cellulose

biodegradation, but it could be underestimated because

anaerobic cellulose hydrolysis is responsible for

considerable carbon recycling in the anoxic zones of

ponds, lakes, oceans, and intestines of ruminants and

guts of termites (P.J. Weimer, personal communica-

tion). Furthermore, an understanding anaerobic cellu-

lase systems are of significant importance to basic

sciences, such as the evolution of cellulase genes, the

structures of cellulases, and the formation and

hydrolysis of reacting biofilms on cellulose surfaces

(Lynd et al., in press). Anaerobic cellulose fermenta-

tion has both current and future applications, such as

agricultural processes anaerobic waste treatment, andconsolidated bioprocessing (CBP), respectively (Lynd,

1996; Lynd et al., 1999, 2002, 2005). Recently, a

microbial cellulose hydrolysis mechanism has been

reported for the anaerobic cellulolytic bacterium

Clostridium thermocellum that assimilates longer

soluble hydrolysis products with an average degree

of polymerization of 4 rather than glucose and

cellobiose. The improved bioenergetics resulting

from longer chain sugar assimilation supports the

biological feasibility of anaerobic fermentation without

added saccharolytic enzymes (Zhang and Lynd,2005c). More information about the cellulosome-

based microbial cellulose hydrolysis research is

available elsewhere (Lynd, 1996; Lynd et al., 2002,

1999, 2005; Zhang and Lynd, 2003a, 2004a, 2005a).

3. Substrates for cellulase activity assays

Substrates for cellulase activity assays can be divided

into two categories, based on their solubility in water

(Table 2).

Table 2

Substrates containing -1,4-glucosidic bonds hydrolyzed by cellulases and their detections

Substrate Detection a Enzymes

Soluble

Short chain (low DP)

Cellodextrins RS, HPLC; TLC Endo, Exo, BG

Radio-labeled cellodextrins TLC plus liquid scintillation Endo, Exo, BG

Cellodextrin derivatives

-methylumbelliferyl-oligosaccharides Fluorophore liberation, TLC Endo, Exo, BG

p-nitrophenol-oligosaccharides Chromophore liberation, TLC Endo, Exo, BG

Long chain cellulose derivatives

Carboxymethyl cellulose (CMC), hydroxyethyl cellulose (HEC) RS; viscosity EndoDyed CMC Dye liberation Endo

Insoluble

Crystalline cellulose-

Cotton, microcrystalline cellulose (Avicel), RS, TSS, HPLC Total, Endo, Exo

Valonia cellulose, bacterial cellulose RS, TSS, HPLC

Amorphous cellulose - PASC, alkali-swollen cellulose RAC RS, TSS, HPLC, TLC Total, Endo, Exo

Dyed cellulose Dye liberation Total, Endo

Fluorescent cellulose Fluorophore liberation Total

Chromogenic and fluorephoric derivatives

Trinitrophenyl-carboxymethylcellulose (TNP-CMC) Chromophore liberation Endo

Fluram-cellulose Fluorophore liberation Endo, Total

Practical cellulose-containing substrates

-cellulose, pretreated lignocellulosic biomass HPLC, RS Total

a RS, reducing sugars; TSS, total soluble sugars.



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3.1. Soluble substrates

Soluble substrates include low DP cellodextrins from

2 to 6 sugar units and their derivatives, as well as long

DP cellulose derivatives (ca. several hundreds of sugar

units). They are often used for measuring individualcellulase component activity (Table 2).

Cellodextrins are soluble for DP6, and very

slightly soluble for 6


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accessibility to cellulase that can be estimated based on

maximum cellulase adsorption (Zhang and Lynd,

2004b).

The crystallinity index (CrI) of cellulose, quantita-

tively measured from its wide range X-ray diffraction

pattern (Krassig, 1993; Ramos et al., 2005; Zhang andLynd, 2004b), is not strongly associated with hydrolysis

rates (Converse, 1993; Mansfield et al., 1999; Zhang

and Lynd, 2004b). Nevertheless, it is still a convenient

indicator representing the change in cellulose character-

istics for one material before and after treatment. Cotton,

bacterial cellulose, and the Valonia ventricosa algal

cellulose are examples of highly crystalline cellulose

(Boisset et al., 1999; Fierobe et al., 2002), whereas

amorphous cellulose is at the other extreme. Microcrys-

talline cellulose, filter paper,-cellulose, and pretreated

cellulosic substrates have modest CrI values, and can beregarded as a combination of crystalline fraction and

amorphous fraction, but there is no clear borderline

between two fractions.

Cotton fiber is made from natural cotton after

impurities, such as wax, pectin, and colored matter,

have been removed (Wood, 1988). Whatman No.1

filter paper is made from long fiber cotton pulp with a

low CrI=45% (Dong et al., 1998; Henrissat et al.,

1985). Microcrystalline cellulose, called hydrocellulose

or avicel (the commercial name), can be purchased

from several companies, such as FMC, Merck, and

Sigma. It is made through the following steps:hydrolysis of wood pulp by dilute hydrochloric acid

to remove the amorphous cellulose fraction, formation

of colloidal dispersions by high shear fields, followed

by spray drying of the washed pulp slurry (Fleming et

al. , 2001; Zhang and Lynd, 2004b). However,

microcrystalline cellulose still contains a significant

fraction of amorphous cellulose. Avicel is a good

substrate for exoglucanase activity assay, because it

has a low DP and relatively low accessibility (i.e., the

highest ratio of FRE/Fa) (Table 1). Therefore, some

researchers feel that avicelase activity is equivalentto exoglucanase activity (Wood and Bhat, 1988).

However, some endoglucanases can release consider-

able reducing sugars from avicel (Zhang and Lynd,

2004b).

Bacterial cellulose (BC) is prepared from the pellicle

produced by Acetobacter xylinum (ATCC 23769)

(Hestrin, 1963) or from Nata de Coco (Daiwa Fine

Produces, Singapore) (Boisset et al., 2000). Bacterial

microcrystalline cellulose (BMCC) can be prepared

from BC by partial acid hydrolysis to remove the

amorphous cellulose fraction, resulting in a reduction in

DP (Valjamae et al., 1999).

Amorphous cellulose is prepared by converting the

crystalline fraction of cellulose to the amorphous form

by mechanical or chemical methods. These celluloses

include mechanically made amorphous cellulose, alkali-

swollen cellulose, and phosphorous acid swollen

cellulose (PASC, Walseth cellulose). Mechanicallymade amorphous cellulose is often prepared by ball

milling or severe blending (Fan et al., 1980; Ghose,

1969; Henrissat et al., 1985; Wood, 1988). Alkali-

swollen amorphous cellulose is made by swelling

cellulose power in a high concentration of NaOH

(e.g., 16% wt/wt) producing the cellulose type II from

type I (O'Sullivan, 1997; Wood, 1988). Phosphoric acid

swollen cellulose (PASC) is most commonly made by

swelling dry cellulose powder by adding 85% o-

phosphoric acid (Walseth, 1952; Wood, 1988). High

concentration phosphoric acid treatment could result insome degree of conversion of type II cellulose from type

I (Weimer et al., 1990). The properties of amorphous

cellulose made by ball milling, NaOH and H3PO4, vary

greatly, depending on cellulose origins, reaction tem-

perature and time, as well as reagent types and

concentrations. Therefore, it is nearly impossible to

compare hydrolysis rates on various types of amorphous

cellulose from different laboratories or even different

batches of amorphous cellulose preparations from the

same laboratory. Amorphous cellulose should be kept in

hydrated condition; simple air-drying dehydration

results in a loss of substrate reactivity (Zhang andLynd, 2004b). The loss of substrate reactivity during

dehydration can be minimized through freeze drying or

drying after solvent exchange (Fan et al., 1981; Lee et

al., 1980).

Regenerated cellulose is often made by converting

insoluble cellulose to soluble form using cellulose

solvents, such as nitric acid, sulfuric acid, ammoniacal

cupric hydroxide (Cu(NH3)4(OH)2), N,N-dimethylace-

tamide (DMAc)/LiCl (Striegel, 1997), and 1-butyl-3-

methylimidazolium Cl (Swatloski et al., 2002), followed

by restoration to physically insoluble form. The majorcommercial regenerated cellulose is viscose rayon,

which is not pure amorphous cellulose due to some re-

crystallization. Regenerated amorphous cellulose (RAC)

can be made by using cold 85% H3PO4 to dissolve

cellulose slurry, followed by precipitation with cold

water. RAC is a very good homogeneous substrate for

cellulase activity assays (Zhang et al., 2006), and is

different from Walseth cellulose, prepared from hetero-

geneous swollen cellulose (Walseth, 1952). RAC has a

consistent quality from batch to batch, and is an ideal

insoluble nonsubstitutation cellulose substrate for mea-

suring extremely low cellulase activity.



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-Cellulose contains major cellulose and a small

amount of hemicellulose. The commercial Sigma -

cellulose is often used as a reference cellulosic material

to evaluate the hydrolysis ability of total cellulase (Kim

et al., 2003). Holocellulose is a solid residue of wood

(lignocellulose) after removal of lignin;-cellulose is asolid residue of holocellulose after removal of major

hemicellulose by alkali extraction (Green, 1963); after

the neutralization of soluble alkali extract materials from

holocellulose, the insoluble fraction and the soluble

fraction are -cellulose and -cellulose, respectively

(Corbett, 1963a; Corbett, 1963b).

Lignocellulose pretreatment breaks up the recalci-

trant structure of lignocellulose so that cellulase can

hydrolyze pretreated lignocellulose faster and more

efficiently. Current leading lignocellulose pretreatment

technologies, including dilute acid, hot water, flowthrough, ammonia fiber explosion (AFEX), ammonia

recycle percolation, and lime, have been recently

reviewed elsewhere (Mosier et al., 2005; Wyman et

al., 2005a,b). In addition, two other pretreatments

steam explosion and organosolvhave been intensively

investigated (Arato et al., 2005; Bura et al., 2002, 2003;

Galbe and Zacchi, 2002; Ohgren et al., 2005; Pan et al.,

2005a,b; Pye and Lora, 1991; Sassner et al., 2005 ;

Soderstrom et al., 2003; Wingren et al., 2003).

The substrate characteristics (e.g., cellulose acces-

sibility, DP, hemicellulose content, and lignin content)

of pretreated lignocelluloses vary greatly, stronglydepending on pretreatment methods and severity, and

on lignocellulose origins. For example, the goal of

AFEX is to break up the linkages among lignin,

hemicellulose, and cellulose, but not to remove any

main component. Therefore, the addition of hemi-

cellulase into the cellulase mixture would be important

for improving overall hydrolysis performance for

AFEX-treated feedstock (Teymouri et al., 2005).

Dilute acid pretreatment not only to breaks the linkage

among lignin, hemicellulose, and cellulose, but also

removes major hemicellulose. Therefore, the additionof hemicellulase is not necessary for an improvement

in cellulase mixture performance; while the addition of

non-hydrolysis proteins (e.g. bovine serum albumin)

into the cellulase mixture could reduce the use of

cellulase because of minimization of non-hydrolysis

adsorption of cellulase to lignin (Pan et al., 2005b;

Wyman CE, personal communication). Organosolv

pretreatment significantly removes both hemicellulose

and lignin (Arato et al., 2005; Pan et al., 2005a; Pye

and Lora, 1991). Therefore, neither hemicellulase nor

other protein blockers need to be added. A novel

cellulose-solvent-based lignocellulose fractionation is

under development by our laboratory; the hydrolysis

rates of residual cellulose samples containing little

hemicellulose and lignin cannot be improved by the

addition of either hemicellulase or non-hydrolysis

protein (Zhang et al., unpublished). In a word,

improvements in the overall performance of cellulasemixture by cocktailing are strongly dependent on

residual lignocellulose properties, and remains in the

trial-and-test stage.

Dyed cellulose is prepared by mixing cellulose

with a variety of dyes, such as Remazol Brilliant Blue

(Holtzapple et al., 1984; Wood, 1988), Reactive

Orange (Gusakov et al., 1985), Reactive Blue 19

(Yamada et al., 2005), and fluorescent dye 5-(4,6-

dichlorotriazinyl) aminofluresceinsm (Helbert et al.,

2003). Because of large variations in the surface areas

of cellulose and the binding conditions, the quantita-tive relationship between released dye and reducing

sugars must be established for each batch of dyed

cellulose.

Insoluble cellulose derivatives, such as slightly

substituted CMC, can be mixed with a variety of dyes,

including Cibacron Blue 3GA and Reactive Orange 14

to produce insoluble dyed-CMC (Ten et al., 2004).

Insoluble cellulose derivatives can also be chemically

substituted with trinitrophenyl groups to produce

chromogenic trinitrophenyl-carboxymethyl cellulose

(TNP-CMC) and fluorophoric Fluram cellulose

(Huang and Tang, 1976). The TNP-CMC has a 25-fold greater sensitivity for endoglucanase activity than

does the reducing sugar dinitrosalicyclic acid method,

and Fluram cellulose gives another 10-fold increase in

sensitivity over TNP-CMC (Huang and Tang, 1976).

However, an increased substitution of TNP-CMC

reduces substrate solubility and impairs cellulase action

along -linked chains (Wood and Bhat, 1988). Some-

times, TNP-CMC is a useful substrate for enzyme

solutions containing reducing agents when the reducing

sugar assay cannot be conducted (Shinmyo et al., 1979).

For example, the cellulosome from the anaerobicbacterium C. thermocellum requires the presence of

reducing agents such as DTT or cysteine for activity

(Johnson et al., 1982a; Morag et al., 1992; Zhang and

Lynd, 2003a).

4. Quantitative assays

All existing cellulase activity assays can be divided

into three types: (1) the accumulation of products

after hydrolysis, (2) the reduction in substrate

quantity, and (3) the change in the physical properties

of substrates.



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4.1. Hydrolysis products

The majority of assays involve the accumulation of

hydrolysis products, including reducing sugars, total

sugars, and chromophores. The most common reducing

sugar assays include the dinitrosalicyclic acid (DNS)method (Ghose, 1987; Miller, 1959), the Nelson-

Somogyi method (Nelson, 1944; Somogyi, 1952), the

2,2-bicinchroninate (BCA) method (Waffenschmidt

and Janeicke, 1987; Zhang and Lynd, 2005b), the 4-

hydroxybenzoylhydrazine (PAHBAH) method (Lever,

1972; Lever et al., 1973), and the ferricyanide methods

(Kidby and Davidson, 1973; Park and Johnson, 1949) in

Table 3. Total soluble sugars, regardless of their chain

lengths, can be measured directly by the phenol-H2SO4method (Dubois et al., 1956; Zhang and Lynd, 2005b) or

the anthrone-H2SO4 method (Roe, 1955; Viles andSilverman, 1949). Glucose can be measured by an

enzymatic glucose kit using coupled hexokinase and

glucose-6-phosphate dehydrogenase (Zhang and Lynd,

2004a), or HPLC after post-hydrolysis conversion to

glucose.

Detection ranges of many sugar assays can be

modified using two strategies: (1) a further dilution

after the color reaction and (2) varying sugar volume per

sample prior to the reaction. For example, the DNS

method was originally designed for 20600 g reducing

sugar per sample (Miller, 1959), but its detection range

can be expanded to samples of 1002500 g, followed

by water dilution (Ghose, 1987). The same is true for the

Nelson-Somogyi method. The Sigma enzymatic glucose

assay kit was designed to measure sugar concentrations

from 200 to 5000 g/L using a reaction mixture

consisting of a 10-L sample plus a 1000-L enzymesolution. However, its detection limits can be lowered to

4100 g/L using a reaction mixture of 500-L sample

plus 500-L 2-fold concentrated enzyme solution

(Zhang and Lynd, 2004a).

Major reducing sugar assays depend on the reduction

of inorganic oxidants such as cupric ions (Cu2+) or

ferricyanide, which accepts electrons from the donating

aldehyde groups of reducing cellulose chain ends. Their

detection ranges vary from less than 1g per sample

to> 2500g per sample (Table 3). The DNS and Nelson-

Somogyi methods are two of the most common assaysfor measuring reducing sugars for cellulase activity

assays because of their relatively high sugar detection

range (i.e., no sample dilution required) and low

interference from cellulase (i.e., no protein removal

required). However, the primary drawback for this

method is the poor stoichiometric relationship between

cellodextrins and the glucose standard (Coward-Kelly et

al., 2003; Ghose, 1987; Kongruang et al., 2004; Wood

and Bhat, 1988; Zhang and Lynd, 2005b). For example,

the results may suffer from an underestimation of

cellulase activity when glucose is used as the standard

Table 3

The common colorimetric sugar assays

Method Sample

(mL)

Reagent

(mL)

G amount

(g/sample)

G concn.

(mg/L)

Ref.

Reducing Sugar Assay

DNS Micro 13 3 20600 6.7600 Miller, 1959

DNS Macro 0.5 3 1002500 2005000 Ghose, 1987

Nelson-Somogyi Micro 15 2 + 2 110 0.210 Somogyi, 1952

Nelson-Somogyi Macro 2 2 + 2 10600 5300 Somogyi, 1952

Nelson Semi-Micro 2 2 5100 2.550 Nelson, 1944

Ferricyanide-1 13 1 + 5 19 0.39 Park and Johnson, 1949Ferricyanide-2 1 0.25 0.181.8 0.181.8 Kidby and Davidson, 1973

PAHBAH Micro 0.5 1.5 0.55 110 Lever, 1972

PAHBAH Macro 0.01 3 550 5005000 Lever, 1972

BCA 0.5 0.5 0.24.5 0.49 Waffenschmidt and Janeicke, 1987

Modified BCA 1 1 0.49 0.49 Zhang and Lynd, 2005b

Total Sugar Assay

Phenol-H2SO4 1 1 + 5 5100 10100 Dubois et al., 1956;

Zhang and Lynd, 2005b

Anthrone-H2SO4 1 1 + 5 5100 10100 Roe, 1955; Viles and Silverman, 1949

Enzymatic Glucose Assay

Glucose-HK/PGHD kit 0.01 1 250 2005000 Sigma Kit

Glucose-HK/PGHD kit 0.5 0.5 250 4100 Zhang and Lynd, 2004a



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and -glucosidase is not in excess (Breuil and Saddler,

1985a,b; Schwarz et al., 1988). The ferricyanide,

PAHBAH, and BCA methods, having higher sensitivity

to reducing sugar, can detect as little as several

micrograms per sample, but suffer from non-specific

interference from protein.Total carbohydrate assays, including the phenol-

H2SO4 method and the anthrone-H2SO4 method, offer

two obvious advantages as compared with reducing

sugar assays: a strict stoichemetic relationship between

cellodextrins (glucose equivalent) and the glucose

standard, and little or no interference from protein. But

they are limited for application to pure celluloses,

because any carbohydrates and their derivatives can

have strong interference readings. Using an enzymatic

glucose assay kit or HPLC can overcome nonspecific

readings from other sugars, but this requires an extrastepconversion of longer cellodextrins to glucose.

Total loss of substrate can be measured by several

means, such as gravimetry and chemical methods. These

methods are not as popular as those involving product

accumulation because they involve tedious procedures,

such as sample centrifugation or filtration followed by

drying. Gravimetry should be employed with care,

because the standard deviation of this method is strongly

associated with sample weight. For example, two

samples of 1mg and 100mg weighed by an analytical

balance with accuracy of 0.1 mg have 10% and 0.1%

standard deviation, respectively. Chemical methods fordetermining substrate loss include the phenol-H2SO4(Dubois et al., 1956), the anthrone-H2SO4 (Viles and

Silverman, 1949), and the K2Cr2O7H2SO4 methods

(Wood, 1988) for residual cellulose, and quantitative

saccharification for different carbohydrate components

(Ruiz and Ehrman, 1996).

Measurable physical cellulose properties represent-

ing cellulase activity include swollen factor, fiber

strength, structure collapse, turbidity, and viscosity.

Earlier assays, involving measurement of the physical

changes of the residual solid cellulose, are reviewedhere for historical interest. Examples of these assays

include the swelling factor (measured by alkali

uptake) and the reduction in tensile strength of thread

and pulp (Oksanen et al., 2000; Wood, 1975). Typically,

the lack of sensitivity limits the use of these assays,

except on special occasions (Oksanen et al., 2000; Pere

et al., 2001; Wong et al., 2000). For example, Toyama et

al. measured total cellulase activity based on the time

needed to disintegrate a 1 1 cm filter paper square

(Wood, 1988). The turbidometric assay measures a

reduction in the absorbance of particle suspension

during the hydrolysis process (Enari and Niku-Paavola,

1988; Johnson et al., 1982a,b; Nummi et al., 1981),

which monitors the overall hydrolysis rate over a long

time but does not measure well the initial hydrolysis rate

for individual enzymes. Amorphous cellulose is recom-

mended for turbidometric assays (Enari and Niku-

Paavola, 1988) because crystalline cellulose hydrolysiscould lead to an initial absorbance increase (Zhang,

unpublished).

Viscosimetric determinations have been used as an

assay for the initial hydrolysis rate for endoglucanases

using soluble cellulose derivatives (Demeester et al.,

1976; Hulme, 1988; Manning, 1981; Miller et al., 1960).

Application of this method relies on the assumption that

the ratio of viscosity-average molecular weight to

number-average molecular weight should remain con-

stant during the period of the assay, which may be true

only for a short time (Hulme, 1988). This method is alsoexperimentally cumbersome and difficult to automate.

4.2. Cellulase activity assays

The two basic approaches to measuring cellulase

activity are (1) measuring the individual cellulase

(endoglucanases, exoglucanases, and -glucosidases)

activities, and (2) measuring the total cellulase activity.

In general, hydrolase enzyme activities are expressed in

the form of the initial hydrolysis rate for the individual

enzyme component within a short time, or the end-point

hydrolysis for the total enzyme mixture to achieve afixed hydrolysis degree within a given time. For

cellulase activity assays, there is always a gap between

initial cellulase activity assays and final hydrolysis

measurement (Sheehan and Himmel, 1999). To be most

meaningful, individual cellulase component assays

must also be based on a reliable estimation of the

amount of individual enzyme component present in the

assay. This information permits the calculation of

specific activity, i.e., bonds broken per milligram

enzyme per unit time.

4.2.1. Endoglucanases

Endoglucanases cleave intramolecular-1,4-gluco-

sidic linkages randomly, and their activities are often

measured on a soluble high DP cellulose derivative,

such as CMC with the lowest ratio of FRN/Fa(Table 1).

The modes of actions of endoglucanases and exoglu-

canases differ in that endoglucanases decrease the

specific viscosity of CMC significantly with little

hydrolysis due to intramolecular cleavages, whereas

exoglucanases hydrolyze long chains from the ends in a

processive process (Irwin et al., 1993; Teeri, 1997;

Zhang and Lynd, 2004b). Endoglucanase activities can



12/30

be measured based on a reduction in substrate viscosity

and/or an increase in reducing ends determined by a

reducing sugar assay. Because exoglucanases also

increase the number of reducing ends, it is strongly

recommended that endoglucanase activities be mea-

sured by both methods (viscosity and reducing ends).Because the carboxymethyl substitutions on CMC make

some glucosidic bonds less susceptible to enzyme

action, a linear relationship between initial hydrolysis

rates and serially diluted enzyme solutions requires (1)

dilute enzyme preparation, (2) a short incubation period

(e.g., 24min) or a very low enzyme loading, (3) a low

DS CMC, and (4) a sensitive reducing sugar assay.

Many workers agree that the BCA method for reducing

sugar assay is superior to the DNS method (Carcia et al.,

1993). For example, the modified BCA method, which

is conducted at 75 C to avoid -glucosidic bondcleavage during the assay, delivers a strict stoichiometry

for the reducing ends of cellodextrins regardless of sugar

chain lengths (Zhang and Lynd, 2005b) and offers a

much higher sensitivity as shown inTable 3(Zhang and

Lynd, 2005b).

Soluble oligosaccharides and their chromophore-

substituted substrates, such as p-nitrophenyl glucosides

and methylumbelliferyl--D-glucosides, are also used to

measure endoglucanase activities based on the release of

chromophores or the formation of shorter oligosaccha-

ride fragments, which are measured by HPLC or TLC

(Bhat et al., 1990; Claeyssens and Aerts, 1992; vanTilbeurgh and Claeyssens, 1985; Zverlov et al., 2002a,

2002b, 2003, 2005).

Endoglucanase activities can also be easily detected

on agar plates by staining residual polysaccharides

(CMC, cellulose) with various dyes because these dyes

are adsorbed only by long chains of polysaccharides

(Flp and Ponyi, 1997; Hagerman et al., 1985; Jang et

al., 2003; Jung et al., 1998; Kim et al., 2000; Murashima

et al., 2002a; Piontek et al., 1998; Rescigno et al., 1994;

Ten et al., 2004). These methods are semi-quantitative,

and are well suited to monitoring large numbers ofsamples. Precision is limited because of the relationship

between the cleared zone diameters and the logarithm of

enzyme activities. For example, differences in enzyme

activity levels less than 2-fold are difficult to detect by

eye (Sharrock, 1988). Unfortunately, most exoglucanase

activities are not detected by these methods, since the

processive action of exoglucanases is blocked by

carboxymethyl substitutions, which prohibits cellulose

chain from shortening. The lack of efficient exogluca-

nase plate screening method explains some of the

difficulty in detecting exoglucanase genes cloned from

C. thermocellum (Demain et al., 2005).

4.2.2. Exoglucanases

Exoglucanases cleave the accessible ends of cellu-

lose molecules to liberate glucose and cellobiose. T.

reesei cellobiohydrolase (CBH) I and II act on the

reducing and non-reducing cellulose chain ends,

respectively (Teeri, 1997; Teeri et al., 1998; Zhangand Lynd, 2004b). Avicel has been used for measuring

exoglucanase activity because it has the highest ratio of

FNR/Faamong insoluble cellulosic substrates (Table 1).

During chromatographic fractionation of cellulase

mixtures, enzymes with little activity on soluble CMC,

but showing relatively high activity on avicel, are

usually identified as exoglucanases. Unfortunately,

amorphous cellulose and soluble cellodextrins are

substrates for both purified exoglucanases and endoglu-

canases. Therefore, unlike endoglucanases and -

glucosidases, there are no substrates specific forexoglucanases within the cellulase mixtures (Sharrock,

1988; Wood and Bhat, 1988).

Claeyssens and his coworkers (van Tilbeurgh et al.,

1982) found that 4-methylumbelliferyl--D-lactoside

was an effective substrate forT. reesei CBH I, yielding

lactose and phenol as reaction products, but it was not a

substrate for T. reesei CBH II (van Tilbeurgh and

Claeyssens, 1985) and some endoglucanases (van

Tilbeurgh et al., 1982). T. reesei EG I, structurally

homologous to CBH I, also cleaves 4-methylumbelli-

feryl--D-lactoside, yet these enzymes can be differen-

tiated by adding cellobiose, an inhibitor that stronglysuppresses cellobiohydrolase activity (Claeyssens and

Aerts, 1992). T. reesei CBH II does not hydrolyze 4-

methylumbelliferyl--D-aglycones of either glucose or

cellobiose units, but does cleave 4-methylumbelliferyl-

-D-glycosides with longer glucose chains (van Til-

beurgh et al., 1985).

Deshpande et al. (1984)reported a selective assay for

exoglucanases in the presence of endoglucanases and -

glucosidases. This assay is based on the following: (1)

exoglucanase specifically hydrolyzes the aglyconic

bond of p-nitrophenyl--D-cellobioside to yield cello-biose and p-nitrophenol, (2) -glucosidase activity is

inhibited by D-glucono-1,5--lactone (Holtzapple et al.,

1990), and (3) the influence of exoglucanase hydrolysis

activities must be quantified in the assay procedure in

the presence of added purified endoglucanases. How-

ever, this technique has its own limitations: (1) CBH II

activity cannot be measured using p-nitrophenyl--D-

cellobioside, (2) the specific activity of the available

purified endoglucanases may not be representative of all

existing endoglucanases in the mixture, and (3) the

product ratio from endoglucanase actions may be

influenced by the presence of exoglucanases.



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4.2.3. -D-glucosidases

-D-glucosidases hydrolyze soluble cellobiose and

other cellodextrins with a DP up to 6 to produce glucose

in the aqueous phase. The hydrolysis rates decrease

markedly as the substrate DPs increase (Zhang and

Lynd, 2004b). The termcellobiaseis often misleadingdue to this key enzyme's broad substrate specificity

beyond a DP of 2. -D-glucosidases are very amenable

to a wide range of simple sensitive assay methods, based

on colored or fluorescent products released from p-

nitrophenyl -D-1,4-glucopyranoside (Deshpande et al.,

1984; Strobel and Russell, 1987), -naphthyl--D-

glucopyranoside, 6-bromo-2-naphthyl--D-glucopyra-

noside (Polacheck et al., 1987), and 4-methylumbelli-

feryl--D-glucopyranoside (Setlow et al., 2004). Also,

-D-glucosidase activities can be measured using

cellobiose, which is not hydrolyzed by endoglucanasesand exoglucanases, and using longer cellodextrins,

which are hydrolyzed by endoglucanases and exoglu-

canases (Ghose, 1987; Gong et al., 1977; McCarthy et

al., 2004; Zhang and Lynd, 2004b).

4.2.4. Total cellulase

The total cellulase system consists of endoglucanases,

exoglucanases, and -D-glucosidases, all of which

hydrolyze crystalline cellulose synergically. Total cellu-

lase activity assays are always measured using insoluble

substrates, including pure cellulosic substrates such as

Whatman No. 1 filter paper, cotton linter, microcrystal-line cellulose, bacterial cellulose, algal cellulose; and

cellulose-containing substrates such as dyed cellulose,

-cellulose, and pretreated lignocellulose.

The heterogeneity of insoluble cellulose and the

complexity of the cellulase system cause formidable

problems in measuring total cellulase activity. Experi-

mental results show that the heterogeneous structure of

cellulose (filter paper and bacterial cellulose) gives rise

to a rapid decrease in the hydrolysis rate within a short

time (less than an hour), even when the effects of

cellulase deactivation and product inhibition are takeninto account (Valjamae et al., 1998; Zhang et al., 1999).

In an attempt to clarify this situation, a functionally

based model has been developed to demonstrate that the

degree of synergism between endoglucanase and

exoglucanase is influenced by substrate characteristics,

experimental conditions, and enzyme loading/composi-

tion ratio (Zhang and Lynd, in press). This model clearly

suggests the complexity of total cellulase activity assays

and infers that it is nearly impossible to apply the results

of the total cellulase activity assay measured on one

solid substrate to a different solid substrate. This is one

of the reasons that the U.S. DOE-sponsored cellulase

development projects, conducted by Genencor Interna-

tional and Novozymes Biotech, tailored cellulase

mixture performance based only on an identical

sampledilute acid pretreated corn stover substrate

that was prepared in the pilot plant of the National

Renewable Energy Laboratory (Golden, CO) (Knaufand Moniruzzaman, 2004).

The most common total cellulase activity assay is the

filter paper assay (FPA) using Whatman No. 1 filter

paper as the substrate, which was established and

published by the International Union of Pure and

Applied Chemistry (IUPAC) (Ghose, 1987). This

assay requires a fixed amount (2 mg) of glucose released

from a 50-mg sample of filter paper (i.e., 3.6%

hydrolysis of the substrate), which ensures that both

amorphous and crystalline fractions of the substrate are

hydrolyzed. A series of enzyme dilution solutions isrequired to achieve the fixed degree of hydrolysis. The

strong points of this assay are (1) it is based on a widely

available substrate, (2) it uses a substrate that is

moderately susceptible to cellulases, and (3) it is based

on a simple procedure (the removal of residual substrate

is not necessary prior to the addition of the DNS

reagent). However, the FPA is reproduced in most

laboratories with some considerable effort and it has

long been recognized for its complexity and suscepti-

bility to operators' errors (Coward-Kelly et al., 2003;

Decker et al., 2003). Reliability of results could be

influenced by (1) the -D-glucosidase level present inthe cellulase mixture (Breuil and Saddler, 1985a,b;

Schwarz et al., 1988; Sharrock, 1988), because the DNS

readings are strongly influenced by the reducing end

ratio of glucose, cellobiose, and longer cellodextrins

(Ghose, 1987; Kongruang et al., 2004; Wood and Bhat,

1988; Zhang and Lynd, 2005b); (2) the freshness of the

DNS reagent, which is often ignored (Miller, 1959); (3)

the DNS reaction conditions, such as boiling severity,

heat transfer, and reaction time (Coward-Kelly et al.,

2003); (4) the variations in substrate weight based on the

area size (1 6 cm a strip), because this method does notrequire substrate excess (i.e., substrate amounts strongly

influence enzyme activity) (Griffin, 1973); and (5) filter

paper cutting methods, because the different paper-

cutting methods such as paper punching, razoring, or

scissoring could lead to different accessible reducing

ends of the substrate (Zhang and Lynd, 2005b).

Dyed celluloses are widely used for determining

sugar inhibition for total cellulase because they avoid

the high background interference from added sugars

(Gusakov et al., 1985c; Holtzapple et al., 1984; Wood,

1988). Fluorescent-dyed cellulose is also used for the

same purpose, and the higher signal per molecule of

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fluorescent dye permits detection of lower cellulase

activities. Researchers should consider the following:

(1) the calibration curve between dye release and

reducing sugar accumulation should be established for

each batch of substrate, because dye adsorption depends

on cellulosic substrate properties and preparationconditions; (2) the calibration curve works only for a

small hydrolysis conversion range, because dye mole-

cules cannot enter into the internal cellulose structure;

and (3) the different hydrolysis modes of endogluca-

nases and exoglucanases have different dye release

preferences (Helbert et al., 2003). Using dyed cellulose,

Holtzapple et al. (1984) showed that glucose and

cellobiose were noncompetitive inhibitors to the T.

reeseicellulase. On the contrary, theT. longibrachiatum

cellulase was competitively inhibited by cellobiose and

glucose (Gusakov et al., 1985c). Some feel that thedifferent inhibition patterns may be attributed to large

variations in characteristics of dyed celluloses (Gruno et

al., 2004).

Cotton fiber, microcrystalline cellulose, bacterial

cellulose, and algal cellulose are several other common

pure cellulosic substrates. Powder microcrystalline

cellulose could become a preferred substrate to replace

filter paper because (1) it can be rapidly dispensed

volumetrically as a slurry and thus permits robotics

methods; (2) it can be easily pelleted by centrifugation,

and the total sugars released are measured more exactly

by the phenol-H2SO4 method than by the DNS assay;(3) it is a more recalcitrant substrate, yielding a more

stringent substrate for total cellulase activity than does

filter paper; and (4) activities measured on microcrys-

talline cellulose could more accurately represent

hydrolysis ability on pretreated lignocellulose, because

its characteristics are closer to those of pretreated

lignocelluloses, based on cellulose accessibility to

cellulase and the degree of polymerization (Zhang and

Lynd, 2004b). Sigmacell-20, a readily available micro-

crystalline cellulose powder, could also be a good

alternative substrate for a total cellulase activity assay,replacing Whatman No. 1 filter paper. Keep that in

mind, some of the pretreated lignocellulose still contains

significant amounts of hemicellulose and lignin, while

microcrystalline cellulose does not contain hemicellu-

lose and lignin.

-Cellulose and pretreated lignocellulose are often

used to evaluate the digestibility of commercial cellulase

or of a reconstituted cellulase mixture for a prolonged

reaction. The primary difference, as compared to

cellulase activity assays using model cellulosic sub-

strates, is the time required for assays, which ranges

from several minutes to hours for model substrates

(initial hydrolysis rate) to several days for pretreated

lignocellulose to obtain the final digestibility (cellulose

conversion). Clearly, the presence of hemicellulose and

even lignin results in more complexity. Again, the

desired outcome of the experiment must indicate the

substrate chosen, especially in the case of total cellulaseperformance.

In conclusion, the measurement of isolated individ-

ual cellulase activity is relatively easy, but it is still

challenging to measure T. reesei CBH I and CBH II

activities specifically in the presence of endoglucanases.

There is no clear relationship between the hydrolysis

rates obtained on soluble substrates and those on

insoluble substrates, mainly because of huge differences

in substrate accessibility and DP. For insoluble cellu-

lose, it is highly unlikely that any substantial solubili-

zation of crystalline or semicrystalline cellulose willproceed linearly with time, due to varying-glucosidic-

bond accessibilities and chain end availability for

different regions of fibers. Researchers must state

clearly all parameters of their assay conditions, and

resist temptation to compare their results to those of

other researchers using different substrates, assay

methods, etc. For example, the specific activity of

Thermobifida fuscaYX endoglucanase is reported to be

at least ten-fold higher than that of T. reesei endoglu-

canase on soluble CMC (Himmel et al., 1993); however,

this activity ratio is not maintained if the assays are

performed with insoluble cellulose (Himmel et al.,1999).

5. Cellulase improvement and screening/selection

Two strategies are available for improving the

properties of individual cellulase components: (1)

rational design and (2) directed evolution.

5.1. Rational design

Rational design is the earliest approach to proteinengineering, was introduced after the development of

recombinant DNA methods and site-directed mutagen-

esis more than 20years ago, and is still widely used.

This strategy requires detailed knowledge of the protein

structure, of the structural causes of biological catalysis

or structure-based molecular modeling, and of the

ideally structurefunction relationship. As shown in

Fig. 3, the process of rational design involves (1) choice

of a suitable enzyme, (2) identification of the amino acid

sites to be changed, based usually on a high resolution

crystallographic structure, and (3) characterization of

the mutants. The availability of data on the protein



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structure of an enzyme or of homologous proteins

typically governs the choice of a suitable enzyme for

modification. The identification of the region of the

protein to be modified generally requires the knowledgeof not only the existing function of the region but also

the desired modified or new function. The modification

of amino acid sequence can be achieved through site-

directed mutagenesis, exchange of elements of second-

ary structure, and even exchange of whole domains and/

or generation of fusion proteins. The faith in the power

of rational design relies on the belief that our current

scientific knowledge is sufficient to predict function

from structure. But such information of structures and

mechanisms is not available for the vast majority of

enzymes. Even if the structure and catalysis mechanismof the target enzyme are well characterized, the

molecular mutation basis for the desired function may

not be achieved (Arnold, 2001).

Rational design appears to be a logical method for

researchers to examine possible amino acid sites near to

the active site or the binding pocket in a 3-dimensional

structure (Bornscheuer and Pohl, 2001). But many

important enzymatic properties are not localized in a

small number of catalytic residues a priori. Indeed,

many residues distributed over large parts of the protein

often confer important properties. Even when large

functional changes can be obtained with a few amino

acid substitutions, it will often be difficult or impossible

to discern the specific mutations responsible. For

example, a significant increase (106-fold) in the

specificity constant (kcat/KM) of aspartate aminotrans-ferase favoring valine requires 17 amino-acid changes,

only one of which occurs within the active-site

(Benkovic and Mames-Schiffer, 2003). Recently, a

successful computational design to convert non-active

ribose binding protein to triose phosphate isomerase was

based on 1822 mutations and exhibited a 105106 fold

activity enhancement (Dwyer et al., 2004). Unfortu-

nately the success of computational models is often

limited to well-understood reactions and enzymes.

Different from most enzymes catalyzing soluble

substrates in the aqueous phase, cellulase acting oninsoluble heterogeneous cellulose is a more complex

process, involving: (1) the changes in heterogeneous

cellulose characteristics during hydrolysis (Banka et al.,

1998; Boisset et al., 2000; Chanzy et al., 1983; Din et

al., 1991, 1994; Halliwell and Riaz, 1970; Lee et al.,

1996, 2000; Saloheimo et al., 2002; Walker et al., 1990,

1992; Wang et al., 2003; Woodward et al., 1992; Zhang

and Lynd, 2004b); (2) cellulase diffusion, adsorption,

and catalysis on the surface of cellulose, i.e., decreases

from a 3-dimension diffusion (in liquid phase) to a 2-

dimension diffusion (on solid surfaces) (Henis et al.,

1988; Katchalski-Katzir et al., 1985) and even 1-

Protein structure

Structure-based molecular modeling

Site-directed mutagenesis

Characterization of mutants

Transformation and

Expression

Repeat(optional)

Fig. 3. Scheme of rational protein design.



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dimension processivity along cellulose chains for

cellobiohydrolases (Teeri, 1997); (3) the non-productive

cellulase binding on the cellulose surface (Beldman et

al., 1987; Sheehan and Himmel, 1999); and (4) the yet

unexplained dynamic interactions among the cellulose-

binding module (CBM), the catalytic domain, and asingle glucan chain end lifted from the cellulose surface

(Skopec et al., 2003).

Several excellent reviews summarize numerous

studies using site-directed mutagenesis for investigating

cellulase mechanisms and improving enzyme properties

(Schulein, 2000; Wilson, 2004; Wither, 2001). Not

surprisingly, few researchers using site-directed muta-

genesis have reported successful examples of signifi-

cantly higher activity cellulase mutants on insoluble

substrates (Escovar-Kousen et al., 2004; Sakon et al.,

1996; Zhang et al., 2000a,b; Zhang and Wilson, 1997).One clear example, however, is the report by Baker and

coworkers of a 20% improvement in the activity on

microcrystalline cellulose of a modified endoglucanase

Cel5A from Acidothermus cellulolyticus (Baker et al.,

2005). The Cel5A endoglucanase, whose high-resolu-

tion crystallographic structure has been available

(Sakon et al., 1996), was subjected to a series of

mutations designed to alter the chemistry of the

product-leaving side of the active site cleft. Using

structural information and following a thesis that end

product inhibition could be relieved by a substitution of

non-aromatic residue at site 245, a mutant (Y245G) wasshown to increase KI of cellobiose by 15-fold.

However, today there are no general rules for site-

directed mutagenesis strategies for improving cellulase

activity on solid cellulase substrates and it remains in a

trial-and-test process.

5.2. Directed evolution

Our still limited knowledge about the characteristics

of insoluble cellulose substrates, the dynamic interac-

tions between cellulases and insoluble substrates, and

the complex synergetic and/or competitive relationshipsamong cellulase components, significantly limits

rational design for improving cellulase properties,

despite increasing understanding of cellulase structures

and hydrolysis mechanisms, characterization of cellu-

lose properties, and cellulase adsorption (Bothwell et al.,

1997; Bothwell and Walker, 1995; Bourne and Henris-

sat, 2001; Lynd et al., 2002; Wither, 2001; Zhang and

Lynd, 2004b). In 1999, Michael Himmel (Sheehan and

Himmel, 1999) wrote: non-informational approaches

to protein engineering should be used to complement

existing efforts based on informational or rational designstrategies in order to ensure success of the DOE

cellulase improvement program. One approach to

non-informational mutant identification is irrational

design using directed evolution.

The greatest advantage of directed evolution is that it

is independent of knowledge of enzyme structure and of

the interactions between enzyme and substrate. The

greatest challenge of this method is developing tools to

correctly evaluate the performance of mutants generated

by recombinant DNA techniques. The success of a

directed evolution experiment depends greatly on the

method chosen for finding the best mutant enzyme,often stated as you get what you screen for(Hibbert et

al., 2005; Schmidt-Dannert, 2001; Schmidt-Dannert and

Arnold, 1999) (seeFig. 4).

Table 4lists the published examples of the cellulases

with properties altered using directed evolution. Four

Fig. 4. Scheme of directed protein evolution.



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directed evolution examples have been reported for

endoglucanases, all of which are identified by facilitated

screening on solid plates containing CMC, followed by

Congo Red staining (Catcheside et al., 2003; Kim et al.,

2000; Murashima et al., 2002a; Wang et al., 2005).Kim

et al. (2000) reported that a 5-fold higher specific

activity Bacillus subtilis endoglucanase mutant was

found by screening cellulase mutants, generated by

DNA shuffling and displayed on the surface ofE. coli

by fusion of the Pseudomonas syringae ice-nucleationprotein. Doi et al. (Murashima et al., 2002b) enhanced

the thermostability of an endoglucanase by seven-fold

using the family shuffling technique based on the

parental Clostridium cellulosomal endoglucanases-

EngB and EngD. Gao et al. (Wang et al., 2005) found

that aT. reeseiEG III mutant generated using the error-

prone PCR technique and expressed inSaccharyomyces

cerevisiae was found to have an optimal pH of 5.4,

corresponding to a basic pH shift of 0.6. Another

example identified hybrid mutants using the family

shuffling technique forT. reesei cel12A and Hypocreaschweinitzii cel12Agenes (Catcheside et al., 2003).

-D-glucosidase mutants have been reported to be

screened blindly using 96-microplate wells because of

lack of facilitated screening tools (Arrizubieta and

Polaina, 2000; Gonzalez-Blasco et al., 2000; Lebbink

et al., 2000; McCarthy et al., 2004). Improvements in

the low temperature catalysis (3-fold) for the hyperther-

mostable Pyrococcus furiosus -D-glucosidase CelB

(Lebbink et al., 2000) and the thermostabilities and

catalytic efficiencies for the Paenibacillus polymyxa

BgblA and BglA were obtained using the chromogenic

substrate, p-nitrophenyl--D-glucopyranoside (Arrizu-

bieta and Polaina, 2000; Gonzalez-Blasco et al., 2000).

The hydrolysis rate of theThermotoga neapolitana1,4-

-D-glucan -glucohydrolase (GghA) (EC 3.2.1.74)

mutant is increased by 31% after error-prone PCR

mutagenesis, in which blind screening was based on

glucose released from a non-chromogenic substrate

(cellobiose) and measured by the coupled reactions of

thermostable glucokinase and glucose-6-phosphate

dehydrogenase (McCarthy et al., 2004). In another

recent example, after DNA family shuffling, a -glycosidase mutant was found to display lactose

hydrolysis rates 3.5-fold and 8.6-fold higher than the

parental P. furiosus CelB and Sulfolobus solfataricus

LacS, respectively, where glucose released from lactose

was measured using a coupled glucose oxidase and

phenol 4-aminophenazone peroxidase reaction (Kaper

et al., 2002).

In some cases, glycosyl hydrolases, e.g.,Agrobacter-

ium sp. -D-glucosidase, can be converted to glyco-

synthases by site-directed mutagenesis (Mackenzie et

al., 1998). There is no intrinsic way to screen or select forglycosynthase activities today. The specific activity of

glycosynthase fromAgrobacteriumsp. -D-glucosidase

was improved (Kim et al., 2004) using a novel coupled-

enzyme assay and screening on solid plates because

another endoglucanase releases fluorophores from the

fluorogenic product synthesized by glycosynthase

(Mayer et al., 2001). Another selection method for a

glycosynthase mutant library is the chemical comple-

mentation method (Lin et al., 2004), based on the

principle that the glycosynthase activity is linked to the

transcription of a LEU2 reporter gene, resulting in cell

growth dependant on glycosynthase activity. A 5-fold

Table 4

List of cellulases and relevant enzymes whose properties have been changed using directed evolution techniques

Enzyme Altered

property

DNA technique Screening/Selection Ref.

Endoglucanase Thermal

stability

Family shuffling Facilitated screening-Congo red+ CMC agar Murashima et al., 2002b

Endoglucanase Activity DNA shuffling Facilitated screening-Congo red+CMC agar Kim et al., 2000

Endoglucanase Alkali pH epPCR Facilitated screening-Congo red + CMC agar Wang et al., 2005

Endoglucanase Family shuffling Facilitated screening-Congo red+ CMC agar Catcheside et al., 2003

-D-glucosidase Cold adoption DNA shuffling Random Screening-chromogenic substrate Lebbink et al., 2000

-D-glucosidase Thermal

stability

epPCR Random Screening-chromogenic substrate Gonzalez-Blasco et al., 2000

-D-glucosidase Thermal

stability

epPCR+Family

shuffling

Random Screening-chromogenic substrate Arrizubieta and Polaina, 2000

-D-glucosidase Activity epPCR Random Screening-coupled to color reaction McCarthy et al., 2004

-glycosidase Activity Family shuffling Random Screening-chromogenic substrate Kaper et al., 2002

Mutated-glucosidase

(glycosynthase)

Activity epPCR Facilitated Screening-fluorogenic substrate Kim et al., 2004

Mutated endoglucanase

(glycosynthase)

Activity cassette mutegenesis Chemical complementation Lin et al., 2004



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higher activity of glycosynthase is obtained using this

approach (Lin et al., 2004).

Although a number of successful examples using

directed evolution for desired cellulases have been

published, the largest limitation of all current selection

and screening methods is based on soluble substrates.It is still very challenging to design a method to

screen or select cellulase mutants using solid cellulosic

substrates.

5.3. Screening

The screening strategy is a critical step for finding the

desired mutants from a large mutant library. Screening

can be divided into two categories: (1) facilitated

screening, which distinguishes mutants on the basis of

distinct phenotypes, such as chromospheres released orhalos formed, and (2) random screening, which picks

mutants blindly (Taylor et al., 2001).

A typical facilitated screening, carried out on solid

agar, relies on product solubilization followed by an

enzymatic reaction that gives rise to a zone of identity,

such as chromophores released from chromogenic

substrates. The assays may be coupled to a second

enzyme whose product can in turn be easily monitored,

as demonstrated by a successful coupling for cyto-

chrome P450 to horseradish peroxidase (Joo et al.,

1999a,b). With the help of microscopic plate images, it

is feasible to screen a much larger number of clones onsolid plates (e.g., several hundreds per cm2)(Delagrave

et al., 2001; Joo et al., 1999b; Youvan et al., 1995 ).

Recently, an ultra-high throughput facilitated screening

method, based on solid microbeads, has been developed

in which single cells containing mutant genes are

immobilized on solid beads. After a chromogenic

substrate is applied, stronger colored beads containing

desired mutants are identified under the microscope

(Freeman et al., 2004). Another facilitated screening

method, conducted in the liquid phase, applies a flow

cytometer for detecting chromospheres released fromchromogenic substrates, which are catalyzed by the cell-

displayed enzyme. Numerous reviews pertaining to cell

surface displayed enzyme library screening by flow

cytometers are available elsewhere (Aharoni et al.,

2005; Becker et al., 2004; Cohen et al., 2001; Goddard

and Reymond, 2004; Lin and Cornish, 2002; Wahler

and Reymond, 2001; Wittrup, 2001).

Endoglucanase activities are detected easily by

examination of halos on solid agar plates using

CMC as the substrate, followed by Congo Red staining

and washing. Higher hydrolysis rates of mutants usually

result in larger halos (in Section 4.2.1). It is not

surprising that all reported endoglucanase examples

using directed evolution have been screened using the

CMC/Congro Red method (in Section 5.2). It may be

operative to screen exoglucanase mutants on solid plates

using soluble chromogenic substrates, such as nitrophe-

nol-cellobioside. However, it is worth noting that thebest screening methods for endoglucanases and exoglu-

canases, capable of hydrolyzing insoluble cellulose,

must be implemented on insoluble cellulose rather than

on soluble cellulose derivatives.

Random screening is another choice, if facilitated

screening is not available. It is often implemented using

96-well microtiter plates, although some researchers are

moving towards 384-well and higher density plate

formats with the help of accurate, low-volume dispens-

ing instruments (Sundberg, 2000). For example, Diversa

has developed an ultra-throughput screening platform,the Gigamatrix, having 400,000 wells containing only

50 nL of liquid substrate per well (Wolfson, 2005). But

the reformatting of 96-well plates into higher density

requires high assay sensitivity and high evaporation

control. Additional product measurement can be

achieved using HPLC, mass spectrometry, capillary

electrophoresis, or IR-thermography (Wahler and Rey-

mond, 2001).

A number of improved -glycosidase mutants after

random mutagenesis are found using 96-well micro-

plates, as reported in Section 5.2. Recently, in order to

measure total cellulase activity, the FPA has beenminiaturized from a 1.5-ml enzyme solution to 60 L,

which is implemented in a 96-microplate well (Xiao et

al., 2004). Water evaporation from samples is prevented

using a PCR thermocycler having a built-in 105 C hot

lid. Also,Decker and coworkers (2003)have developed

a high throughput cellulase assay system using 96-

microplates equipped in a Cyberlabs C400 robotics deck

with the substrates such as Whatman No. 1 filter paper

disks (0.25in. diameter), Solka-Floc, SigmaCell-20,

Avicel PH101 (FMC, Philadelphia, PA), and cotton

linters (Fluka/Sigma Aldrich). This custom system has amaximum output of 84 samples per day and produces

values that correlate to the traditional FPA. However, no

application of these systems to the screening of higher

activity cellulases has been reported. Considering the

inherent limitations of the FPA (see Section 4.2.4), this

automated approach could be benefited by replacing the

DNS method with the phenol-H2SO4 method because

the latter (1) has a higher sugar sensitivity (Table 3), (2)

is independent of oxygen presence (unlike the DNS

reagent) (Miller, 1959), especially for miniaturization

that has a very high surface/volume ratio, (3) yields a

strict stoichiometric relationship between color



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formation and total soluble sugars released, and (4) is

thus independent of -D-glucosidase levels. Different

from FPA, the recommended method requires centrifu-

gation for soluble sugars and solid cellulose residue

prior to the phenol-sulfuric acid assay.

5.4. Selection

Selection is always preferred over screening because

it has several-order-of-magnitude higher efficiency than

screening (Griffithsa et al., 2004; Olsen et al., 2000;

Otten and Quax, 2005). However, selection requires a

phenotypic functional link between the target gene and

its encoding product that confers selective advantage to

its producer. This method is often implemented based on

the principles of resistance to cytotoxic agents (e.g.,

antibiotics) (Stemmer, 1994a,b) or of complementationof auxotroph (Griffithsa et al., 2004; Jurgens et al.,

2000; Smiley and Benkovic, 1994). Today, selection on

solid media in

Outlook for Cellulase Improvemente (Zhang Y.-h. P. Et Al., 2006)

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